The present invention relates to human CD16-II protein variants, DNA sequences coding for them, their use in therapy and/or in diagnosis of autoimmune diseases and inflammatory illnesses, as well as pharmaceutical compositions comprising them.
CD16, also called Fcxcex3 receptor-III (Fcxcex3R-III), is a low affinity receptor for Immunoglobulin G (IgG). With other receptors of the immunoglobulin Fc portion (Fcxcex3R-I, Fcxcex3R-II, Fcxcex5R-I), CD16 plays an important role in mediating autoimmunity and inflammatory responses.
Studies using monoclonal. antibodies against CD16 have established this receptor""s role in removing immune complexes from circulation and in mediating antibody-dependent cell mediated cellular cytotoxicity (ADCC) (see for example Van de Winkel et al., Immunol. Today, 14, 1993, pp.215-221). The binding of IgG with CD16 elicits NK/LGL cell activation and triggers ADCC. ADCC can be halted in the presence of high levels of soluble CD16.
It has been found (see Mathiot et al., J. Clin. Immunol., 13, (1), 1993, pp. 41-8) that the level of soluble CD16 was significantly decreased in patients with multiple myeloma compared with healthy volunteers. In addition a stage-dependent decrease of soluble CD16 was observed, with a highly significant difference between stage I and stages II+III myeloma patients. Therefore the measurement of soluble CD16 in serum is both a diagnostic and a prognostic marker of myeloma, which can be useful to define and guide novel immunomodulatory therapies of the disease.
It has further been found that CD16 is present in human serum and other body fluids and is elevated at sites of inflammation (see Fleit et al., Blood, 79, (10), 1992, pp. 2721-8).
From Ravetch et al. (J. Exp. Med., 170, 1989, pp. 481-97) it is clear that there are at least two isoforms of human CD16, type 1 and type 2, that can be designated as CD16-I and CD16-II, respectively These two isoforms of CD16 are encoded by two separate but elated genes, NA1 and NA2.
From Scallon et al. (PNAS USA, 86, pp.5079-83, July 1989) it is evident that CD16-I and CD16-II are distinct in both structure and cellular expression. CD16-I is expressed predominantly on the surface of neutrophils and monocytes, whereas CD16-II is expressed predominantly on the surface of macrophages, natural killer cells and large granular lymphocytes (NK/LGL). Furthermore, these two types of CD-16 are associated with the cell surface via two distinct mechanisms: CD16 type I is associated with the cell surface by glycosyl-phosphotidylinositol (GPI) linkage; whereas CD16 type II is anchored on the membrane with about 20 extra amino acids. Furthermore, the N-terminus of the mature CD16 has been investigated and the methionine residue at position 18 was identified as the N-terminal residue of the mature protein. Thus, the initial translation product contains a 17-amino acid signal peptide. The transmembrane region of CD16-II is shown to be from amino acid 209 to 229, whereas CD16-I is reported lacking transmembranal and cytoplasmic domains.
It has been determined that a single amino acid at position 203, Ser, found in isoform I versus Phe, found in type II, determines the membrane anchoring mechanism (see Lanier et al., Science, 246, 1989, pp. 1611-3).
For human CD16-I, a polymorphism has been reported previously, as is evident from FIG. 1, whereas only one alternative nucleic acid sequence encoding CD16-II has been published until now (Ravetch et al., J. Exp. Med., 170, 1989, pp. 481-97).
Recently, Huizinga et al. (see Blood, 76, pp. 1927-, 1990) published evidence that CD16-I deficiency is related to neonatal isoimmune neutropenia.
Bredius et al. (in Immunology, 83, pp. 624-, 1994) reported specifically that CD16-I-NA1 exhibited a 21-25% higher IgG1 mediated phagocytosis than CD16-I-NA2.
It has been reported that circulating levels of soluble CD16 are reduced in Multiple Myeloma, and an inhibitory effect of sCD16 on myeloma cells and pokeweed mitogen (PWM)-induced B-cell proliferation have been reported (see, respectively, Hoover et al., J. Cli. Inve., 95(1), pp.241-7, 1995) and Teillaud et al., Blood, 82(10), 15 November 1993).
European Patent Application EP 343 950 generally discloses soluble and membrane-bound human Fcxcex3R-III polypeptides as well as nucleic acids capable of encoding the same. In particular, the .sequence of a CD16-I variant and the sequence of CD16-II are shown in the Figures. This patent application further discloses various utilities for these polypeptides.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.
The present invention is based on the discovery of new human CD16-II variant clones. They have been isolated by using an RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction)-based strategy using designed isoform-specific oligonucleotide primers. In particular, from a pooled human lung RNA extract, CD16-II has been amplified via RT-PCR. These CD16-II variants provide a therapeutic intervening approach and/or a diagnostic tool for autoimmune and inflammatory diseases. As they are natural variants of the CD16-II sequence previously published, the polypeptides of the present invention can be used for any of the utilities previously disclosed for CD16-II. All of the utilities for CD16-II made evident from any of the publications disclosed herein are hereby incorporated herein by reference, and particularly those in European application 343,950.
The main object of the present invention are the polypeptides comprising respectively the SEQ ID NO: 1, 2, 3 and 4.
Another object of the invention are the DNA molecules comprising the DNA sequences coding for each of the four polypeptides, as shown in FIG. 3, including nucleotide sequences substantially the same. xe2x80x9cNucleotide sequences substantially the samexe2x80x9d includes all other nucleic acid sequences which, by virtue of the degeneracy of the genetic code, also code for the given amino acid sequences. Preparation of an alternative nucleotide sequence encoding the same polypeptide but differing from the natural sequence due to changes permitted by the known degeneracy of the genetic code, can be achieved by site-specific mutagenesis of DNA that encodes an earlier prepared variant or a nonvariant version of the polypeptide of the present invention. Site-specific mutagenesis allows the production of variants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 complementing nucleotides on each side of the sequence being altered. In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by publications such as Adelman et al., DNA, 2:183 (1983), the disclosure of which is incorporated herein by reference. As will be appreciated, the site-specific mutagenesis technique typically employs a phage vector that exists in both a single-stranded and double-stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage, for example, as disclosed by Messing et al., Third Cleveland Symposium on Macromolecules and Recombinant DNA, A. Walton, editor, Elsevier, Amsterdam (1981), the disclosure of which is incorporated herein by reference. These phage are readily available commercially and their use is generally well known to those skilled in the art. Alternatively, plasmid vectors that contain a single-stranded phage origin of replication (Veira et al., Meth. Enzymol., 153:3 (1987)) may be employed to obtain single-stranded DNA. In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the relevant protein. An oligonucleotide primer bearing the desired mutated sequence is prepared synthetically by automated DNA/oligonucleotide synthesis. This primer is then annealed with the single-stranded protein-sequence-containing vector, and subjected to DNA-polymerizing enzymes such as E. coli polymerase I Klenow fragment, to complete the synthesis of the mutation-bearing strand. Thus, a mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli JM101 cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
As already stated, the proteins of the invention are useful in the therapy and/or diagnosis of autoimmune diseases and inflammatory illnesses. Therefore, in a further aspect, the present invention provides the use of each protein of the invention in the manufacture of a medicament for the treatment of autoimmune diseases and inflammatory illnesses.
The medicament is preferably presented in the form of a pharmaceutical composition comprising one of the proteins of the invention together with one or more pharmaceutically acceptable carriers and/or excipients. Such pharmaceutical compositions form yet a further aspect of the present invention.
The invention will now be described by means of the following Example, which should hot be construed as in any way limiting the present invention. The Example will refer to the Figures specified here below.